The invention relates to human high affinity choline transporter polynucleotides, polypeptides and their uses.
The cholinergic transmissions or neuromodulations in the central nervous system are involved in a number of fundamental brain processes such as learning and memory (Aigner and Mishkin, Behav. and Neural. Biol. 45:81-87 (1986); Fibinger, TINS, 14:220-223 (1991)), arousal, and sleep-wake cycles (Karczmar, in Biology of Cholinergic Function, (eds A. M. Goldberg and I. Hanin) Raven Press, N.Y. 1976, pp 395-449; Fibinger, 1991). In this system, the formation of the neurotransmitter acetylcholine is catalyzed by the enzyme choline acetyltransferase (ChAT, E.C. 2.3.1.6), which transfers an acetyl group from acetylcoenzyme A to choline, in the presynaptic nerve terminals of cholinergic neurons. Acetylcholine is packaged into the synaptic vesicles by a vesicular acetylcholine transporter (VAChT) and is then ready to be released in a calcium dependent manner. Acetylcholine binds specifically to either the nicotinic or muscarinic receptors (AChR) to transmit information to the postsynaptic neurons. The action of acetylcholine is terminated through hydrolysis to acetate and choline by the enzyme acetylcholinesterase (ACHE, E.C.3.1.1.7). Most of the choline is then transported back to the presynaptic terminal to be recycled as one of the precursors for the biosynthesis of acetylcholine. This step, which is mediated by the action of the high affinity choline transporter (HACT), is believed to be the rate limiting step of the biosynthesis of the neurotransmitter acetylcholine, which plays a pivotal role in processes such as learning, memory, and sleep (Karczmar, 1976; Figinger, 1991).
Altered functioning of the cholinergic system has been observed during normal aging processes (Cohen et al., JAMA, 274:902-7 (1995); Smith et al., Neurobiol Aging, 16:161-73 (1995)), while its dysfunction underlies nicotine addiction and a number of neurological and psychiatric disorders most notably Alzheimer""s disease (AD), Myasthenia Gravis, Amyotrophic Lateral Sclerosis (ALS), and epilepsies. Clearly, molecular cloning of these cholinergic components are important in the understanding of the cholinergic mechanism and neurotransmission in the central nervous system during normal aging processes and under certain disease states such as AD and ALS. So far, all the components but one have been cloned. The cloned molecules include biosynthetic enzyme choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), both muscarinic and nicotinic type acetylcholine receptors (AChR), and acetylcholinesterase (AChE). The availability of these agents or tools has advanced cholinergic research tremendously which has led to more insights and knowledge on how cholinergic systems function. However, the sodium-dependent high affinity choline transporter (HACT), which is believed to be the rate limiting step for the neurotransmitter biosynthesis and regeneration has not yet been cloned. In order to obtain a more thorough understanding about how the cholinergic mechanism operates in the central nervous system and how cholinergic neurotransmissions and modulations are regulated at the molecular level, the high affinity choline transporter must be cloned.
In the cholinergic nerve synaptosome preparations, two types of choline uptake systems have been described (Yamamura and Snyder, J. Neurochem. 21:1355-1374 (1973); Haga and Noda, Biochem. Biophys. Acta, 291:564-575 (1973); Jope, Brain Res. Rev. 1, 313-344 (1979)), one of which uses sodium as electrogenic driving force and is of high affinity for choline with an apparent Km less than 10 xcexcM (HACT). Choline transporting activity of this type of uptake system is associated with efficient conversion of choline to acetylcholine and choline transporting activity is inhibited by low concentrations of hemicholinium-3 (Ki=25 to 100 nM). While the other uptake system does not depend on sodium, it exhibits a lower affinity for choline with an apparent Km between 40 to 100 xcexcM (low affinity choline transporter, LACT), and is hemicholinium-3 insensitive. Studies over the last two decades indicate that the high affinity choline uptake system is coupled either physically (Barker and Mittag, J. Pharmacol. Exp. Ther. 192:86-94 (1975)) or kinetically (Jope and Jenden, Life Sci. 20:1398-92 (1977)) to the biosynthesis of the neurotransmitter acetylcholine. In vivo pharmacological studies conducted by Kuhar and his colleagues suggest that the high affinity choline transporter may play a regulatory role in addition to the rate limiting step in acetylcholine biosynthesis (Kuhar and Murrin, J. Neurochem. 30:15-21 (1978)). Phospholipase A2 and cAMP pathways were reported to act synergistically to regulate high affinity choline transporter activity (Cancela et al., Biochem. Biophys. Res. Commun. 213:944-949 (1995); Vogelsberg et al., J. Neurochem. 68:1062-70 (1997)).
Recently, age-related alterations in the density of cholinergic reuptake sites were examined in discrete brain regions of behaviorally tested rats using autoradiography. A strong correlation was found between behavioral performance of aged rats and density of the binding sites for hemicholinium-3 in dorsal hippocampal subfield CA3 and dentate gyrus (Smith et al., Neurobiol Aging. 16:161-73 (1995)). Similarly, a 3 to 4 fold decreased brain choline uptake in normal, older human adults was reported using an in vivo proton magnetic resonance spectroscopy, indicating uptake of circulating choline into the brain decreases with age (Cohen et al., JAMA, 274:902-7 (1995)). Interestingly, an increase in high affinity choline transport was observed in the cortical brain region of Alzheimer""s patients, suggesting disordered regulation of this rate limiting component of acetylcholine synthesis is above and beyond that required to compensate for the reduced cholinergic synaptic functionality (Bissette et al., Ann. N.Y. Acad. Sci., 777:197-204 (1996)). Undoubtedly, isolation of the high affinity choline transporter gene would allow the mechanism of transport to be studied at the molecular level and could provide further insights to its function and regulation under normal and pathological conditions.
So far, attempts using various strategies to purify and isolate a high affinity choline transporter gene have not been successful (for a recent review on this topic, see Happe and Murrin, J. Neurochem. 60:1191-1201 (1993)). Isolation and purification of a transporter molecule using biochemical methods have been difficult partly due to fact that the transporter is present in low amounts and becomes unstable in later stages of purification (Rylett, J. Neurochem. 51:1942-5 (1988)). Limited biochemical characterization of HACT has revealed proteins with molecular sizes of 42, 58, and 90 kDa, which were labeled with tritiated choline mustard aziridinium ion from a Torpedo electric organ membrane preparation (Rylett, 1988). An 80 kDa protein from locust head ganglia has been labeled with tritiated hemicholinium-3 and isolated by usage of a monoclonal antibody that blocks HACT activity (Knipper et al., FEBS Lett. 245:235-237 (1989); Knipper, Neurochem. Int. 14:211-215(1989)). The polypeptide is capable of accumulating choline into liposomes, is hemicholinium-3 sensitive, and has the same ionic and energy requirements as HACT from other sources (Knipper et al., Biochem. Biophys. Acta, 1065:107-113 (1991)). Further purification yields a protein with an apparent molecular size of 90 kDa which becomes a 65 kDa protein upon treatment by endoglycosidase F (Knipper et al., 1991). These results suggest that at least, in the locust, the HACT molecule is a single polypeptide, although whether the functional HACT requires multiple subunits is not clear.
A second approach of cloning HACT using an Xenopus oocyte expression system has been explored by several laboratories (O""Regan et al., Mol. Brain Res. 32:135-42 (1994)). Hemicholinium-3 sensitive HACT activity could be induced upon introduction of fractionated mRNAs from Torpedo electric lobe tissues (O""Regan et al., 1994). However, a truncated synaptotagnin C2 domain was found to be responsible for this HACT activity while full length synaptotagmin is not capable of this action (O""Regan et al., 1994). It is not clear what this finding means, nonetheless, it did point out that the endogenous choline transporter activity in Xenopus oocyte could obscure the signal generated by the cDNAs. Another approach based on homology cloning strategy has also been attempted without success. In the last six years, several neurotransmitter transporters have been cloned, including those for norepinephrine, dopamine, serotonin, gama-aminobutyric acid (GABA), glycine, and proline (Amara and Kuhar, Annu. Rev. Neurosci. 16:73-94 (1993); Malandro and Kilberg, Annu. Rev. Biochem. 65:305-36 (1996)). All of the transporters appear to belong to a single family of proteins of approximately 600-700 amino acids and have 12 membrane spanning domains. One would anticipate HACT to be one member of this family although it is possible that a unique or a significantly different structure is required by HACT. Homology cloning of a choline transporter based on the conserved regions of this family was claimed, but later this molecule was identified as the creatine transporter (Mayser et al., FEBS Lett. 305:31-36 (1992). Thus it appears a search for alternative approach to clone HACT is fully warranted and desirable.
It is an object of the invention to provide human high affinity choline transporter polynucleotides and polypeptides and compositions comprising human high affinity choline transporter polynucleotides and polypeptides. It is another object of the invention to provide vectors and recombinant host cells comprising human high affinity choline transporter polynucleotides and polypeptides. It is a further object of the invention to provide methods for identifying test agents that decrease or increase high affinity choline transporter protein activity and for identifying binding partners of high affinity choline transporter polypeptides. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides an isolated high affinity choline transporter protein which has the amino acid sequence shown in SEQ ID NO:2, and naturally occurring biologically active variants thereof.
Another embodiment of the invention provides an isolated polypeptide that comprises about at least six contiguous amino acids of a high affinity choline transporter as shown in SEQ ID NO:2.
A further embodiment of the invention provides a composition comprising a high affinity choline transporter protein as shown in SEQ ID NO:2 or a polypeptide comprising about at least six contiguous amino acids of a high affinity choline transporter as shown in SEQ ID NO:2 and a pharmaceutically acceptable carrier. The composition can optionally comprise a choline acetyltransferase polypeptide.
Still another embodiment of the invention provides an antibody or antigen binding portion thereof that specifically binds to a high affinity choline transporter polypeptide as shown in SEQ ID NO:2.
Yet another embodiment of the invention provides an isolated polynucleotide which encodes a high affinity choline transporter protein as shown in SEQ ID NO:2. The polynucleotide can comprise the sequence shown in SEQ ID NO:1. The polynucleotide can be present in a vector, and the vector can in turn be present in a recombinant host cell.
Even another embodiment of the invention provides a polynucleotide comprising about at least 12 contiguous nucleic acids of a polynucleotide as shown in SEQ ID NO:1.
Another embodiment of the invention provides a composition comprising a polynucleotide which encodes a high affinity choline transporter polypeptide as shown in SEQ ID NO:2 or a polynucleotide that comprises the sequence shown in SEQ ID NO:1 and a pharmaceutically acceptable carrier.
Still another embodiment of the invention provides a method of screening test agents for the ability to decrease or increase high affinity choline transporter protein activity. A first host cell comprising about at least 12 contiguous nucleic acids of a polynucleotide as shown in SEQ ID NO:1 is contacted with a test agent and choline. A second host cell comprising about at least 12 contiguous nucleic acids of a polynucleotide as shown in SEQ ID NO:1 is contacted with choline. The amount of choline that is transported into the first cell and the second cell is measured and the amounts of choline transported into the first cell and into the second cell are compared. A test agent that decreases the amount of choline transported into the first cell as compared to the second cell is a potential drug for decreasing high affinity choline transporter protein activity, and a test agent that increases the amount of choline transported into the first cell as compared to the second cell is a potential drug for increasing high affinity choline transporter protein activity.
Yet another embodiment of the invention provides a method for identifying a binding partner of a polypeptide that comprises about at least six contiguous amino acids of a high affinity choline transporter as shown in SEQ ID NO:2. The polypeptide is contacted with a candidate binding partner. Whether the binding partner binds to the polypeptide is determined.
The invention therefore provides for the first time human HACT polynucleotides, polypeptides, compositions, and uses thereof.